This application is submitted in response to PAR #03-063. Supplemental funds are requested for grant # U01 HL69748 for studies with human embryonic stem (hES) cells. The goals of this supplement are (1) to develop capabilities in the growth and differentiation of hES cells, and (2) to transplant CD34+hematopoietic precursors differentiated from a hES cell line expressing the enhanced green fluorescent protein (EGFP) as a marker gene. We propose to use an established hES cell line (UC06; HSF-6) listed on the NIH Stem Cell Registry which has previously been transduced using an HIV-l-derived lentiviral vector (HIV/VSV-MND-EGFP) to generate CD34+ cells for fetal monkey transplant purposes. This will allow the assessment of the safety of transplant of CD34+EGFP+ cells differentiated from a hES cell line in a primate model, and the study of human hematopoiesis in the rhesus host. Initial cell culture experiments will determine the optimal time point in the development of CD34+ cells from embryoid bodies (EBs) and analysis by flow cytometry (Specific Aim 1). Cells will be grown in culture and assessed on days 3, 4, 5, and 6 to identify the day on which the largest quantity of CD34+ cells are present. BMP-4 will be added to half of the hES cell cultures to determine if this approach increases the frequency of the desired phenotype. CD34+EGFP+ cells will be collected and cryopreserved using a controlled rate freezing protocol in preparation for fetal transplant (Specific Aim 2). Using established ultrasound-guided techniques, we will transplant CD34+ cells differentiated from hES cells into first trimester fetal monkeys using an intraperitoneal approach. Fetuses will be monitored sonographically then delivered at term, and cells of donor origin identified by flow cytometry, immunoselection, laser-capture microdissection, immunohistochemistry, and PCR-based assays. While hES cells hold great human clinical promise, the potential for tumor formation, uncontrolled expansion, and/or rejection post-transplant requires rigorous linvestigation in nonhuman primates before considering the use of these cells in humans. One of the goals of the Center for Fetal Monkey Gene Transfer is to advance the field of gene therapy for future human application, and this supplemental request will aid in accomplishing this goal. [unreadable] [unreadable]